The transplantation of major categories of central nervous system (CNS) cells (i.e. neurons, astrocytes) or CNS tissue fragments offers opportunities to study the developmental biology and immunological properties of these cells, to create animal models of CNS diseases and injuries and to develop alternative strategies for the treatment of spinal cord injuries and progressive neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and hereditary ataxia as well as to study other diseases, conditions and disorders characterized by loss, damage or dysfunction of neurons including transplantation of neuron cells into individuals to treat individuals suspected of suffering from such diseases, conditions and disorders. Indeed, recent pioneering efforts to utilize human fetal mesencephalic tissue grafts to ameliorate the extrapyramidal manifestations of drug induced and idiopathic Parkinson's disease emphasize the potential of transplanted human CNS tissues for the treatment of human neurodegenerative diseases (Freed, C. A., et al. 1992 New Engl. J. Med. 327:1549-1555; Spencer, D. D. et al. 1992 New Engl. J. Med. 327:1541-1548; and Widner, H., et al. 1992 New Engl. J. Med. 327:1556-1563). However, the results of these efforts have not been completely satisfactory.
The immortalization of CNS progenitor cells using constructs containing temperature sensitive promoters has enabled transplantation of genetically engineered precursors of neurons and glia, but brain grafts of these progenitors have given rise to mixed populations of glial and neuronal progeny (Cattaneo, E., and R. McKay 1991 TINS 14:338-340; Renfranz, P. J., et al. 1991 Cell 66:713-729; Snyder, E. Y., et al. 1992 Cell 68:33-51). An alternative strategy has been to use neuron-like transformed cell lines obtained from tumors of the CNS, but neoplastic neuron-like cells usually cannot be induced to permanently exit the cell cycle or they develop into tumors when transplanted into the rodent brain (Fung, K.-F. et al. 1992 J. Histochem. Cytochem. 40:1319-1328; Trojanowski, J. Q., et al. 1992 Molec. Chem. Neuropathol. 17:121-135; and Wiestler, O. D. et al. 1992 Brain Pathol. 2:47-59). A slowly dividing human neuronal cell line obtained from a child with unilateral megalencephaly was shown to exhibit a neuron-like phenotype in culture but grafts of these cells in the rodent CNS showed a mixture of neuronal and mesenchymal phenotypic properties (Poltorak, M., et al. 1992 Cell Transplant I:3-15).
Thus, regeneration of injured spinal cord or brain tissue has been an elusive goal for many years. Over 250,000 Americans are spinal cord injured, with 15,000 new injuries reported each year. More than half of them were injured between the ages of 16 and 30, with the majority (90%) of people surviving and living near normal life spans. So far, medicine has improved the quality of care and life for those with spinal cord injuries. However, modern care is expensive, in some cases reaching $1.35 million per person and as much as $4 million per institutionalized patient.
The cells of the spinal cord form a complex circuit which underlies the transmission of sensory information centrally and motor commands peripherally. The complex processing required for the execution of such intricate behaviors is reflected in the complexity of the types of neurons in the spinal cord. The many morphological subtypes of neurons collectively express most known neurotransmitters, neuromodulators and receptors, such as serotonin (5-HT), noradrenaline, glycine, acetylcholine, GABA and glutamate. Often many transmitters and modulators are present within the same cell. For example, serotonergic fiber neurons have been shown to co-express thyrotropin releasing hormone (TRH), 5-HT and substance P in the same terminals (Shapiro, S., 1997, Neurosurgery, 40, 168-177). Replacement of the phenotypic variation normally present in the spinal cord is therefore a central goal of therapeutic research.
Numerous therapies have been tried over the years. For example, U.S. Pat. No. 4,966,144 discloses a method of transplanting a nerve graft into a transected site of the spinal cord and irradiating the site with low energy light. The nerve graft is a peripheral nerve segment or spinal cord segment and is placed in the injured area so that its longitudinal axis is parallel to that of the spinal cord.
U.S. Pat. No. 5,639,618 discloses a stable line of lineage-specific neuronal stem cells. The stem cells are constructed from blastocyst-derived ES cells transfected with a reporter construct under the control of the Otx regulatory region, Otx being an early marker for neurogenesis. The reporter construct is used to segregate the neuronal stem cells by FACS isolation or other methods. The segregated cells are then plated and permitted to terminally differentiate.
U.S. Pat. No. 5,618,531 discloses a method for increasing the viability of cells which are administered to the brain or spinal cord. The method is accomplished by attaching the cells to a support matrix and implanting the support matrix into the brain.
U.S. Pat. No. 5,135,956 discloses using long-chain (23 to 29 carbons) fatty alcohols and prodrug esters to cause extension of neurites in vivo and facilitate healing of traumatic injury to both the central and peripheral nervous systems by facilitating reconnection and reestablishment of function, decreasing ischemia and neuronal death, and reducing Wallerian degeneration after injury.
Xenotransplantation, the use of cells from different species, has also been suggested as a viable approach to circumventing the limitations associated with human fetal neural transplantation (Galpern W R, et al. 1996 Experimental Neurology 140:1-13). Transplant of porcine cells harvested from the midbrains of pig fetuses is currently being evaluated in clinical trials.
Encapsulated xenografts of rat PC12 cells that secrete dopamine have also been developed. A semipermeable polymer membrane allows diffusion of the small therapeutic molecules but prevents diffusion of the larger immunogenic molecules. Whether the release of dopamine from encapsulated sources will be sufficient to restore optimal dopamine levels in Parkinson's Disease patients remains to be determined.
However, while cells derived from non-human animals are potential candidates for human neural transplantation, they carry the risks of transferring intrinsic pathogens, creating novel infectious agents, or eliciting deleterious immune responses (Isacson, O. and Breakefield X. 1997 Nature Medicine 3:964-969).
According to Anton et al. (1994 Exp. Neurol. 127:207-218), the ideal cell for a CNS transplant system should meet the following criteria: It should be of human CNS origin, capable of growth cessation and differentiation, clonal and defined, transfectable and selectable, immunologically inert, capable of long-term survival following implantation, non-tumorigenic, functional and integrated into the host brain, of consistent quality, and readily available.
The present invention provides neuronal cell transplants useful in studying and treating spinal cord and brain injuries and neurodegenerative disorders.